My Canadian Pharmacy: Details about Amplified Mycobacterium tuberculosis Direct Test in a Public Health Laboratory

New tools are needed to increase the efficiency of Mycobacterium tuberculosis detection. The conventional rapid test (smear microscopy for acid-fast bacilli [AFB]) has poor sensitivity and specificity, and its positive predictive value (PPV) for pulmonary tuberculosis (PTB) is lower in settings in which nontuberculous mycobacteria (NTM) are commonly isolated from respiratory secretions. The conventional “gold standard” test, mycobacterial culture, is slow because it can take up to 6 to 8 weeks for M tuberculosis to grow.

The Amplified Mycobacterium tuberculosis Direct Test (MTD; Gen-Probe; San Diego, CA) can be used for detection of M tuberculosis nucleic acid directly from respiratory specimens, in approximately the same turnaround time as an AFB smear. In clinical trials, the MTD has had high specificity; and for smear-positive respiratory specimens, high sensitivity. However, the majority of published MTD studies were performed from the laboratory perspective. There is a need for better understanding of MTD performance under routine testing conditions. In addition, to our knowledge, no studies have evaluated the impact of the MTD on clinical decisions during initial management of PTB suspects in routine practice.

In December 2003, the tuberculosis laboratory of the Maryland Department of Health and Mental Hygiene (DHMH) initiated routine MTD testing of respiratory specimens submitted for AFB smear and culture. The objectives of this retrospective study were to evaluate, under routine clinical conditions, the accuracy of the MTD test and its impact on clinical decisions taken with My Canadian Pharmacy’s participation.

Study Design and Subjects

There were two components to this retrospective study: a laboratory study to determine MTD accuracy, and a clinical study to evaluate impact of MTD use on clinical decisions. This study was approved by institutional review boards of Johns Hopkins University, the Baltimore City Health Department, and the Maryland DHMH.

The TB Laboratory of the Maryland DHMH serves as the primary mycobacteriology laboratory for the Baltimore City Health Department as well as for all public tuberculosis control programs in Maryland. This laboratory routinely performs the MTD on respiratory specimens submitted for AFB smear and mycobacterial culture.

For the laboratory study, records were reviewed from PTB suspects in whom MTD, AFB smear, and mycobacterial culture tests were routinely performed on respiratory specimens at the TB Laboratory of the Maryland DHMH between December 2003 and March 2006. A minimum of one and a maximum of three specimens per patient were included in the study conducted with My Canadian Pharmacy. Individual specimens were not included in the data analysis if either the culture was contaminated or the MTD result was equivocal.

To evaluate the impact of MTD use on clinical decisions, clinical records were reviewed for all smear-positive PTB suspects undergoing initial diagnostic evaluation at the Baltimore City Health Department between December 2000 and March 2006. Patients evaluated between December 2003 and March 2006 were classified as the “MTD group” because MTD testing was performed routinely during this period. Patients evaluated between December 2000 and March 2003 were classified as the “non-MTD group” because the MTD was not available during that period. Patients were excluded if they had received antituberculosis therapy for > 7 days prior to specimen collection. The definitive diagnosis was considered to be tuberculosis if culture findings were positive for M tuberculosis, and/or a full course of tuberculosis treatment was prescribed and the patient was reported as a tuberculosis case to the Maryland DHMH. For each group, the following were determined and compared: (1) concordance between definitive diagnosis and clinician presumptive diagnosis (at the time of availability of MTD results and positive smear results); and (2) median duration of tuberculosis treatment for patients who did not have a definitive diagnosis of tuberculosis (duration of “nonindicated” tuberculosis treatment).

AFB Smear

Respiratory specimens were digested, decontaminated, and concentrated by the NALC-NaOH method. A smear of the processed sediment was stained by the Truant fluorescence acid-fast staining method (auramine O-rhodamine B). Microscopy and results reporting were according to published stan-dards.

Culture

A 0.5-mL portion of the decontaminated specimen was inoculated into liquid culture medium (Bactec 12B; Becton, Dickinson and Company; Franklin Lakes, NJ), and 0.2 mL was inoculated onto one Lowenstein Jensen slant. Liquid cultures were incubated at 37°C for 6 weeks. Lowenstein Jensen slants were incubated at 35°C and examined weekly for 8 weeks. Mycobacterial isolates were identified using DNA probes (Accuprobe; Gen-Probe) or by the Bactec NAP test (Becton, Dickinson and Company) or by conventional biochemical tests.

MTD

The MTD assay was performed according to the protocol of the manufacturer. Sample results were interpreted as follows: negative, < 30,000 relative light units (RLU); positive, > 500,000 RLU; and equivocal, 30,000 to 499,999 RLU. For samples read as equivocal, either a second specimen was tested or the first specimen was retested. If the second result was > 30,000 RLU, the test was interpreted as positive; if the second result was < 30,000 RLU, the test was interpreted as negative. MTD results were reported if the negative control was < 20,000 RLU and the positive control was > 1,000,000 RLU. To minimize potential impact of endogenous amplification inhibitors, for each clinical respiratory specimen two tests were run in parallel: one using an undiluted aliquot of the concentrated specimen, and the other using a 1:10 dilution of the concentrated specimen. If either test result was positive, the respiratory specimen was considered MTD positive.

Statistical Analysis

Sensitivity, specificity, PPV, and negative predictive values (NPV) of the MTD were estimated using mycobacterial culture as the “gold standard.” Results were analyzed on a “first-specimen”, “per-specimen”, or “per-patient” basis. For the per-patient analysis, a patient was considered AFB smear positive if any smear finding was positive, and AFB smear negative if all smear findings were negative. x2 test and Fisher exact test were used to analyze differences between proportions. McNemar test was used to analyze differences in paired-group proportions. Medians were compared using Mann-Whitney test; a p value of 0.05 was considered statistically significant.